title: Clinical validation of a next-generation sequencing screen for mutational hotspots in 46 cancer-related genes.
abstract: Transfer of next-generation sequencing technology to a Clinical Laboratory Improvement Amendments-certified laboratory requires vigorous validation. Herein, we validated a next-generation sequencing screen interrogating 740 mutational hotspots in 46 cancer-related genes using the Ion Torrent AmpliSeq cancer panel and Ion Torrent Personal Genome Machine (IT-PGM). Ten nanograms of FFPE DNA was used as template to amplify mutation hotspot regions of 46 genes in 70 solid tumor samples, including 22 archival specimens with known mutations and 48 specimens sequenced in parallel with alternate sequencing platforms. In the archival specimens, the IT-PGM detected expected nucleotide substitutions (n = 29) and four of six insertions/deletions; in parallel, 66 variants were detected. These variants, except a single nucleotide substitution, were confirmed by alternate platforms. Repeated sequencing of progressively diluted DNA from two cancer cell lines with known mutations demonstrated reliable sensitivity at 10% variant frequency for single nucleotide variants with high intrarun and inter-run reproducibility. Manual library preparation yielded relatively superior sequencing performance compared with the automated Ion Torrent OneTouch system. Overall, the IT-PGM platform with the ability to multiplex and simultaneously sequence multiple patient samples using low amounts of FFPE DNA was specific and sensitive for single nucleotide variant mutation analysis and can be incorporated easily into the clinical laboratory for routine testing.
authors.affiliation: Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, Texas, USA.
authors.forename: Rajesh R
authors.initials: RR
authors.lastname: Singh
authors.forename: Keyur P
authors.initials: KP
authors.lastname: Patel
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Routbort
authors.forename: Neelima G
authors.initials: NG
authors.lastname: Reddy
authors.forename: Bedia A
authors.initials: BA
authors.lastname: Barkoh
authors.forename: Brian
authors.initials: B
authors.lastname: Handal
authors.forename: Rashmi
authors.initials: R
authors.lastname: Kanagal-Shamanna
authors.forename: Wesley O
authors.initials: WO
authors.lastname: Greaves
authors.forename: L Jeffrey
authors.initials: LJ
authors.lastname: Medeiros
authors.forename: Kenneth D
authors.initials: KD
authors.lastname: Aldape
authors.forename: Rajyalakshmi
authors.initials: R
authors.lastname: Luthra
mesh_terms: D014644:Genetic Variation; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006801:Humans; D016254:Mutagenesis, Insertional; D009154:Mutation; D009369:Neoplasms / Q000175:diagnosis* / Q000235:genetics*; D009857:Oncogenes; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion